Malaria treatments

ABSTRACT

PCT No. PCT/GB93/00084 Sec. 371 Date Dec. 5, 1994 Sec. 102(e) Date Dec. 5, 1994 PCT Filed Jan. 15, 1993 PCT Pub. No. WO93/15761 PCT Pub. Date Aug. 19, 1993A method of treating or preventing clinical manifestations associated with diseases caused by infectious organisms which express antigens which in the patient stimulate secretion of harmful levels of at least one cytokine, other than diseases caused by organisms which stimulate secretion of cytokines only by expression of lipopolysaccharide, which method comprises administering to a human in need thereof an effective non-toxic amount of a material selected from the group consisting of inhibitors and immunogens wherein said inhibitors are pharmacologically acceptable materials which, in vitro, reduce or abolish secretion, by at least one of human monocytes and mouse peritoneal macrophages, of tumour necrosis factor following stimulation with a phospholipid-containing; tumour necrosis factor-inducing antigen other than lipopolysaccharide, and wherein said immunogens are pharmacologically acceptable materials comprising at least one B-cell epitope and which stimulate production of antibodies which, in vitro, reduce or abolish secretion, by at least one of human monocytes and mouse peritoneal macrophages, of tumour necrosis factor following stimulation with a phospolipid-containing, tumour necrosis factor-inducing antigen other than lipopolysaccharide. Examples of inhibitors include inositol monophosphate and phosphatidyl inositol lipids. Immunogens include these inhibitors optionally with carrier proteins.

This application is a 371 of PCT/GB93/00084 filed Jan. 1, 1993.

The present invention relates to the treatment and prevention of malariaand certain other infectious diseases, especially to materials for usein such treatment and prevention.

Malaria is caused by organisms of the genus Plasmodium which infect andmultiply within erythrocytes. Blood-stage infection is usuallycharacterised by severe fever, sometimes accompanied by anaemia,hypoglycemia, pulmonary oedema, renal or hepatic failure, and coma whichmay occasionally prove fatal. Immunity may develop so as to reduce theseverity of infection but takes many years and in individuals living inendemic areas complete elimination of parasites rarely occurs.Nevertheless, children from these areas do appear to develop resistanceto the above clinical manifestations of infection even while carryingheavy parasite loads.

The conventional approaches to controlling malaria have aimed at:

(a) preventing the spread of the disease by eliminating the mosquitovector or

(b) killing or controlling the parasite by chemotherapy. Insecticideshave not been effective in controlling the mosquito due to the rapiddevelopment of resistance and also present environmental difficultiesrendering this approach unsatisfactory. Chemotherapy has been relativelyeffective but is expensive; there are dangers to the patient if theparasites are killed too rapidly and supportive measures are generallyrequired to deal with the major effects of the disease, such ashypoglycaemia which is treated by administration of glucose.Experimental vaccines aimed at eliminating one or other stages of theparasite are under trial but do not seem to protect all individuals.None of these control measures have been fully successful and thedisease is on the increase world wide. Protection can be provided byvarious drugs for those travelling to endemic regions, but this is not apractical solution for the whole at-risk population.

Various investigators have observed that the major clinical problemsraised by episodes of malarial disease are associated with theover-secretion of tumour necrosis factor (TNF), and possibly, othercytokines and that secretion of cytokines by macrophages can bestimulated in vitro by malaria antigens. This originally led the presentinventors and their collaborators to postulate that antigens whichstimulated cytokine release by macrophages would be particularly usefulin generating anti-disease immunity against malaria Playfair et al.Immunology Today, 1990, 11, 25!, resulting in work on "antigen 7"published in, for instance, PCT/DK90/00159 (WO-A-90/15621). It has nowbeen appreciated that these antigens could themselves induce many of theclinical problems associated with malaria and that they would beunlikely to be acceptable for use in humans. Others have proposed thatsecretion of TNF and other cytokines might be prevented by variousagents acting directly to destroy or disable the macrophages, or haveattempted to remove circulating cytokines from the bloodstream, forinstance using anti-TNF antibodies. However both techniques havedisadvantages in that they might compromise aspects of the patient'simmune response to this and other diseases. Moreover a patient'smacrophage population rapidly recovers from the action of direct-actingagents and this makes it unlikely to be practical to treat malaria inthis manner.

On the basis of further investigations the present inventors believethat TNF and other cytokines are secreted by sensitive macrophages as adirect result of binding of malaria antigens to specific receptors onthe macrophages; by interrupting this binding they consider that it ispossible to treat or prevent the clinical manifestations of malaria. Thestrategy avoids the disadvantages of techniques used or proposed byothers and would afford control of the disease while allowing the immunesystem of the patient to control the infection. It is not important thatTNF should be the only or even the main cytokine induced by the diseaseorganism, only that the organisms produce antigens which stimulate therelevant receptors on sensitive macrophages

Following this theory, the inventors have identified certain features ofthe cytokine-stimulating antigens and the receptor enabling them (a) toproduce inhibitors of the binding which may be used prophylactically ortherapeutically against the disease and (b) to select immunogens whichmay be used to stimulate antibody production in at-risk and infectedindividuals so as to protect against the clinical manifestations of thedisease. Furthermore, this insight has led the inventors to the viewthat certain other diseases, in which antigen binding to the samereceptors, and corresponding release of cytokines, plays a leading rolein causing the associated clinical manifestations, may be treated orprevented in similar manner. An example is sleeping sickness caused byAfrican trypanosomes.

Gram negative bacteria also produce TNF-inducing antigens, known aslipopolysaccharide or LPS antigens, but it appears that these act atdifferent receptors such that the inhibitors of the present invention donot significantly affect the course of these bacterial diseases.Similarly, agents intended to prevent LPS-induced TNF secretion havelittle effect on the receptors involved in oversecretion of cytokinesinduced by malarial antigens. The present invention is thereforeconcerned with treatment of diseases caused by infectious organismswhich express antigens which stimulate harmful levels of TNF and/orother cytokines other than diseases caused by organisms which stimulateTNF and/or other cytokine secretion via LPS and the LPS receptor.Accordingly the following description will refer to the diseases to betreated as "non-LPS diseases" and to the cytokine-inducing antigensinvolved as "non-LPS antigens". The organisms involved are hereafterreferred to as "non-LPS organisms" for consistency of terminology but itshould be noted that certain non-LPS organisms which cause disease bythe non-LPS mediated stimulation of cytokine oversecretion may alsoexpress LPS antigens.

The inventors have further observed that inhibitors of theantigen:receptor binding not only prevent the over-secretion of TNF andother cytokines but also have a rapid effect in countering thehypoglycaemia commonly associated with bouts of malaria. This effect onhypoglycaemia is not mediated by insulin and may therefore be achievedby a separate mechanism.

The present invention will be described below with reference to thefigures of the accompanying drawings in which:

A BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows: the inhibition of TNF induction by P. yoelii exoantigensby detoxified exoantigen Legend: Macrophages were incubated with serialdilution of detoxified preparation mixed with a single concentration ofthe active supernatant.

A. Exoantigen dephosphosphorylated by HF treatment.

A representative experiment showing inhibition of an active preparationthat stimulated the production of 102,400 U per ml of TNF by incubationin the presence of a detoxified preparation.

B Exoantigen detoxified by lipase digestion. Means±SD obtained with 3different active preparations incubated in the presence of onedetoxified preparation.

FIG. 2 shows the effect of phosphatidyl inositol, inositol monophosphateand inositol on induction of TNF by P.yoelii exoantigens.

Legend: Preparations of P. yoelii exoantigens that induced theproduction of 18-36,000 U per ml of TNF were incubated with differentconcentrations of PI (∘), IMF () and inositol (□); the results oftypical experiments are illustrated. (The MW of the preparations of PIand IMP used are so similar that the figure would look no different ifplotted in terms of molar concentrations).

FIG. 3 shows: titration of P.yoelii exoantigens in the presence andabsence of inositol monophosphate.

Legend: Yields of TNF were determined from macrophages incubated withserial dilutions of an active supernatant made in medium (∘) or mediumcontaining 20 μg per ml of IMP ().

FIG. 4 shows: the yields of TNF obtained from macrophages stimulatedwith LPS or P. yoelii exoantigens in the presence and absence ofphosphatidyl inositol or inositol monophosphate. Legend: Black-bars=LPS(0.1 μg-1 μg per ml). Hatched bars=exoantigens. Results are means from 3experiments with LPS and 14 with the exoantigens. FIG. 5 shows: thecross-inhibition of TNF induction by exoantigens of human parasitesusing detoxified P.yoelii exoantigens.

Legend: Inhibition of TNF production in response to exoantigens of P.falciparum () and P. vivax (∘) by addition of serial dilutions of alipase-digested P. yoelii supernatant. (Means±SD of 5 experiments for P.falciparum and 3 for P. vivax).

FIG. 6 shows the inhibitory effects of phosphatidyl inositol andinositol monophosphate on TNF induction by exoantigens of the humanparasites.

Legend: Macrophages were incubated with a single concentration of theexoantigens and serial dilutions of the inhibitors. Means (±SD) of atleast 3 experiments.

A: P. falciparum

B: P. vivax

Phosphatidyl inositol (∘) Inositol monophosphate ().

FIG. 7 shows the effect of pretreatment of macrophages on TNF inductionby P.yoelii exoantigens.

Legend: Macrophages were pretreated for 30 mins with the reagents shown,washed, and then stimulated for 1 hr with different preparations of theexoantigens. They were then washed again, incubated overnight and TNFproduction assayed. PI and IMP were used at 20 μg per ml PE and PC at 50μg per ml. The results with detoxified antigens represent 1 of 2experiments; those with the phospholipids and IMP are means (±SD) of 6.

FIG. 8 shows the inhibitory effect of a PAF inhibitor on TNF inductionby P.yoelii exoantigens and LPS.

Legend: Macrophages were incubated overnight with a 1/20 dilution of anexoantigen preparation or 0.2 μg per ml of LPS and differentconcentrations of the inhibitor. Pretreatment of the macrophages with 50μg per ml of inhibitor was not toxic to the cells as the yields of TNFwere unaffected. For legends to FIGS. 9 to 16, see Example 2. Forlegends to FIGS. 17 to 19, see Example 3.

FIG. 9

Inhibition of TNF induction by toxic antigens from P. falciparum and P.yoelii by antisera against IMP assayed using human PBMNC () and mouseperitoneal macrophages (∘) respectively. Means (±SD) of 2 batches ofantiserum, each tested twice against P. falciparum, and of 3 batches(including one of the above) tested against P. Voelii, two of them threetimes and one once.

FIG. 10

Inhibition of TNF induction by parasite antigens by antisera againstPI-KLH.

A: Comparative titrations of antisera obtained after 1 (∘) or 3injections () of PI-KLH. Means (±SD) from 3 different batches ofantiserum, each of which was titrated twice against P. yoelii and P.falciparum antigens.

B: Comparative titrations of affinity purified IgG and IgM fromantiserum obtained after 3 injections of PI-KLH. Means of titrations of2 preparations titrated against P. yoelii antigens that induced 38,956U/ml of TNF from mouse macrophages. Original antiserum (∘) IgG (); IgM(▪)

C: Titrations of antiserum obtained after 3 injections of PI-KLH beforeand after adsorption with various liposomes. Means of titrations of 2serum samples titrated against P. yoelii antigens which stimulated theproduction of 56,000 U/ml of TNF from mouse macrophages. Liposomes usedfor adsorption: none (∘); PC (); PS; (▪) PI (□);

FIG. 11

Inhibition of TNF induction by parasite antigens by antisera againstPS-KLH.

A: Comparative titrations of antisera obtained after 1 () or 3injections (∘) of PS-KLII. Means (±SD) from 5 different batches ofantiserum, titrated against P. yoelii or P. falciparum antigens.

B: Comparative titrations of affinity purified IgG and IgM fromantiserum obtained after 3 injections of PS-KLH. Three different batchesof serum were cooled before adsorption to isotype specific agarose; theywere tested for their ability to inhibit TNF secretion from mousemacrophages stimulated with P. yoelii antigens.

Original antiserum (∘); IgG (); IgM (▪)

C: Titrations of antiserum obtained after 3 injections of PS-KLH beforeand after adsorption with various liposomes. Means of titrations of 2serum samples titrated against P. yoelii antigens which induced theproduction of 13,975 U/ml of TNF from mouse macrophages. Liposomes usedfor adsorption: none (∘); PC () PS (▪); PI (□).

FIG. 12

Prolongation of production of inhibitory antibody by immunisation withPI-KLH or PS-KLH. Pools of serum from groups of 3 mice bled at differentintervals after 3 injections of PI-KLH, PS-KLH, unconjugated PI or P.falciparum antigens were titrated against P. falciparum antigens whichinduced the production of the order of 1,000 pg/ml of TNF from humanPBMNC. PI-KLH (); PS-KLH (∘); PI (□); P. falciparum antigens (▪).

FIG. 13

Inhibition of TNF induction by parasite antigens by antisera againstphospho-threonine and galactosamine-1-phosphate conjugated to KLH.compared with antisera against PI-KLH and PS-KLH Mice were bled 12 daysafter 3 injections in each case and one group immunized with lysineconjugated to KLH is included as a control. Titrations were done againsteither P. falciparum or P. yoelii antigens and are the means of resultsobtained with antisera raised against 2 preparations each ofphospho-threonine-KLH and galactosamine-1-phosphate-KLH, 3 of PI-KLH, 5of PS-KLH and 3 of lysine-KLH. P-threonine-KLH (▪);galactosamine-1-P-KLH (□); PI-KLH (); PS-KLH (∘); lysine-KLH (Δ)

FIG. 14

Titrations of antiserum against phospho-threonine-KLH andgalactosamine-1-phosphate-KLH before and after adsorption with variousliposomes.

A: Phospho-threonine-KLH; B: Galactosamine-1-phosphate-KLH. Means of 2different antisera in each case, titrated for their ability to inhibitthe induction of TNF by mouse peritoneal cells stimulated with P. yoeliiantigens. Liposomes used for adsorption: none (); PC (∘); PI (□), PS(▪).

FIG. 15

The effect of antiserum against KLH conjugates on the induction of TNFsecretion by other stimulants. Various stimulants were incubated in thepresence or absence of antiserum against PI-KLH (black bars) or againstPS-KLH (hatched bars) diluted 1/1500 and the amount of TNF secreted byPBMNC was determined. The results are means (±SD) of at least 4 tests,expressed as a percentage of the control without antibody.

FIG. 16

Induction of hypoglycaemia by parasite toxic antigens injected into miceimmunized with various KLH conjugates. Means (±SD) of blood glucose ofgroups of 3 mice (6 for PS/KLH) injected i.p. with 0.5 ml of apreparation of P. yoelii antigens 12 days after 3 injections of theimmunogens. Unimmunized (▪); lysine-KH (□); PI-KLH (∘); PS-KLH ();P-Thr-KLH (Δ). Although the numbers in each group are small, in total 12mice injected with phosphorylated compounds gave significantly differentresults from the 6 controls at 4 and 8 hr (p=<0.0001 by Students Ttest). Similar results were obtained in other experiments using outbredmice immunized with PI conjugated to BSA, PS-BSA and Gal-1-P-BSA.

EXAMPLE 3

Protection of mice by immunization. FIGS. 17 to 19 show results ofexperiments done with the lethal P. yoelli which killed all the controlmice.

FIG. 17 illustrates protection induced by an untreated parasite culturesupernatant, compared with a potent immunizing lysate that giveanti-parasitic protection as opposed to the anti-disease protection weare interested in. It was published in our paper in Immunology Today1990, 11, 25. The figure was based on about 4 mice. It was shown to makethe point that this kind of immunisation is different from that of ananti-parasitic vaccine, in that the mice that survive do so for aprolonged period with a very high parasitaemia.

FIG. 18 illustrates the prolonged survival obtained in experiments inwhich mice were immunised with parasite supernatants which had beenfirst boiled, then protease-treated and then deaminated (with nitrousacid). All these preparations were still active in vitro in terms oftheir ability to stimulate macrophages to secrete TNF. The mice wereimmunized with 0.5 ml of supernatant i.p. and infected 10 days later. Wehave pooled the results of several experiments so that the figurerepresents at least 20 mice per group. It is rare for mice to die afterday 18, although the odd one did.

FIG. 19 illustrates the prolonged survival obtained with mice immunisedwith inositol monophosate (200 μgi.p.) or 2 mg of preparations ofvarious liposomes. Again, we have pooled results to put up the numbersper group and again only the odd mouse died after day 18.

Inhibitors

In one aspect, the invention relates to inhibitors of TNF secretionwhich may be used therapeutically or prophylactically to treat orprevent the clinical manifestations of malarial disease and othernon-LPS diseases, to the production and use of such inhibitors and topharmaceutical compositions of them.

An inhibitor of the present invention is defined as a pharmacologicallyacceptable material which, in vitro, reduces or abolishes TNF secretionby human monocytes or mouse peritoneal macrophages following stimulationwith phospholipid-containing TNF-inducing non-LPS antigens.

The inhibitors of the invention are effective because they prevent orreduce the binding of the non-LPS antigen(s) produced by the pathogenicorganism to the relevant receptor. This can be achieved in one of twoways: either the inhibitor binds to the receptor or it binds to theantigen. In each case it is necessary for the inhibitor to bind in sucha way that it will hinder binding between the relevant portions of thereceptor and the antigen and the simplest way to achieve this is to usematerials which mimic structural features of the antigen or receptorbinding sites.

One particular class of inhibitors of the invention comprises materialswhich mimic a portion of the antigen and can therefore bind to thereceptor but which lack all the structures of the antigen required toinduce TNF or cytokine secretion. Known compounds which fulfil thesecriteria are inositol monophosphate (IMP) and certain phosphatidylinositol (PI) lipids. PI lipids may bear one or two fatty acid sidechains such as palmitoyl, stearyl and lauryl groups. Other compoundswhich may be used in this manner include phosphatidyl serine (PS) andphosphatidyl threonine (PThr) lipids and phosphorylated sugars such asmannose, glucose, galactose and fructose but not glucosamine. Othermaterials which are candidates for use as such inhibitors includedegradation products of non-LPS TNF-inducing malaria antigens or otherparasite non-LPS exoantigens whose TNF-inducing activity has beenabolished. The exoantigens in question may be related to membrane-boundantigens having a glycosyl phosphatidyl inositol (GPI) anchor or similarstructure which fulfils the same membrane-binding function.

GPI anchors are found on many protein or polysaccharide antigens(whether or not associated with malaria and other protozoal parasitediseases) and comprise a carbohydrate moiety linking the antigen to aglucosamine group which is, in turn, bound to one of the inositolcarbons of a PI lipid molecule.

Any protozoal non-LPS antigen which in vitro induces TNF-secretion byhuman monocytes or mouse peritoneal macrophages and whose TNF-inducingactivity can be reduced by chemical or enzymatic treatment is apotential source of inhibitors according to the present invention.Before use as an inhibitor the antigen must be treated so as to reduceor abolish the TNF-inducing activity which may be achieved, for example,by suitable degradation reactions. Degradation techniques which may beemployed include lipase digestion, deacylation, dephosphorylation andphospholipase C digestion such as described in Example 1.

The strain or species of parasite from which the exoantigen is derivedis not particularly important and it is therefore conceivable that, forexample, malarial antigen degradation products and trypanosomal antigendegradation products could each be used to treat both malaria andsleeping sickness.

Another class of inhibitors of the invention comprises materials whichmimic a portion of the binding site on the receptor and which cantherefore bind to the antigen. Such materials would include synthetic,semisynthetic or recombinant receptor molecules or fragments thereofbearing the binding site or receptor molecules or fragments thereofobtained by extraction and purification from suitable monocytes orperitoneal macrophages. Techniques for producing such materials arewithin the ability and knowledge of those skilled in the art.

A further class of inhibitors of the invention comprises antibodies andfragments thereof, such as F(ab')₂ and Fab fragments, single domainantibodies and recombinant antibody-like polypeptides. These antibodiesand fragments thereof bear a suitable antigen-binding site which iscapable of binding to the relevant receptor molecule on monocytes andperitoneal macrophages or to the TNF-inducing antigen of the non-LPSpathogen in such a way as to prevent or reduce binding between theTNF-inducing antigen and the receptor. It should be noted that suchantibodies and fragments need not be directed against the actual bindingsite of the TNF-inducing antigen or its receptor; provided that theantibody or fragment is sufficiently bulky and binds to the antigen orreceptor sufficiently close to the binding site thereof, steric effectswill diminish the binding of the antigen to the receptor. Techniques forproducing such antibodies and fragments are within the ability andknowledge of those skilled in the art.

Yet another class of inhibitors according to the present inventioncomprises the serum components known to bind to phospholipids such asβ2-glycoprotein-1 (also known as apoprotein-H) and low and high densitylipoproteins (LDL and HDL) and C-reactive protein.

The inhibitors and antibodies or fragments thereof according to theinvention may be used in conventional manner as prophylactic ortherapeutic agents. Administration of antibodies or fragments thereofmay be regarded as passive immunisation. Active immunisation isdescribed below in relation to immunogens of the invention.

The present invention therefore provides the use of an inhibitor ashereinbefore defined in the preparation of a medicament for use in thetreatment or prevention of clinical manifestations associated withinfection by a TNF-inducing non-LPS organism.

The invention further provides a method of treatment of an individualinfected with or at risk of infection with a non-LPS organism byadministering a non-toxic amount of an inhibitor as hereinbefore definedsufficient to treat or prevent clinical manifestations associated withsaid infection.

The invention further provides an inhibitor as hereinbefore definedother than IMP and PI lipids for use in a method of treatment of thehuman or animal body by therapy, especially to treat or prevent clinicalmanifestations associated with infection with a non-LPS organism.

The invention further provides an inhibitor as hereinbefore definedwhich is a degradation product of a TNF-inducing non-LPS antigen-of anon-LPS organism.

In each of the above it is preferred that the non-LPS organism is aprotozoal parasite such as Trypanosoma gambiense or T. rhodesiense or aplasmodial parasite, more preferably a plasmodial parasite and mostpreferably Plasmodium falciparum, P. vivax, P. malaria or P. ovale.

Preferably the clinical manifestations which are treated or preventedinclude hypoglycaemia and/or anaemia and/or fever. Preferred inhibitorsaccording to the present invention comprise the delipidated, deacylated,dephosphorylated or otherwise deactivated degradation products ofphospholipid dependent TNF inducing malarial antigens, IMP anddipalmitoyl, dilauryl and distearyl phosphatidyl inositol.

The inhibitors of the present invention may be used as the pure compoundbut are preferably administered in the form of a pharmaceuticalcomposition.

The present invention therefore provides a pharmaceutical compositionfor treating or preventing the clinical manifestations of infection by anon-LPS organism comprising an inhibitor as hereinbefore defined and apharmaceutically acceptable carrier or diluent therefor.

Suitable carriers and diluents will depend upon the chosen route ofadministration and other criteria well known to pharmacists. Foradministration as tablets and powders the diluent or carrier may be anysuitable inert binding or filling agent. For administration as liquidsthe diluent or carrier will be a liquid such as water for injection,demineralised water or a non-aqueous liquid such as an oil. Thecompositions may also contain accessory ingredients such asantioxidants, antimicrobial agents, buffers and, especially forinjectable compositions, agents to adjust the tonicity of thecomposition.

The compositions may be administered by any suitable route such asorally or parenterally for instance by intravenous, intranasal,intramuscular or subcutaneous injection or infusion. As the objectivewill often be to provide whole-body treatment and prevention of clinicalmanifestations and as the presence of substantial quantities of theinhibitor may be required to maintain protection from the disease,administration by continuous infusion may be required or the use ofdepots and sustained release formulations should be considered.

The dosage administered should be sufficient to maintain protection fromnon-LPS antigens without deleterious side effects and can be ascertainedby typical dose ranging experiments. The dosage will of course dependalso on the size, weight, age and state of health of the recipient andthe clearance rate of the inhibitor, the way the inhibitor isdistributed around the body and its relative affinity and avidity forthe macrophage receptor compared with that of the non-LPS antigens whoseeffect is to be countered. However it is presently contemplated that forIMP doses in the region of 1 to 100 mg/kg, would be administered atintervals of from a few hours to twice or even once daily and that theaverage daily dose for a normal adult would be in the range of from 1 to1000 mg; for instance at least 10 mg.

Treatment using the inhibitors according to the present invention may becombined with other therapeutic or prophylactic measures. For instance,the inhibitors of the invention may be used to prevent adverse effectsarising from destruction of the parasites using anti-parasitic agents.Alternatively, the inhibitors may be used in controlling the worsteffects of an acute episode of a parasite infection in combination withother support measures such as treatment for hypoglycaemia and anaemia.

Immunogens

Whereas the inhibitors of the invention are used prophylactically ortherapeutically to interrupt the binding of non-LPS antigens to therelevant receptor, immunogens are used to generate active immunity inindividuals at risk. Immunogens of the invention are also useful ingenerating antibodies in host animals and thus for providing polyclonal,monoclonal or recombinant antibodies and fragments thereof for use asinhibitors of the invention.

The immunogens of the invention will be pharmacologically acceptablematerials capable of stimulating production of antibodies whichantibodies reduce or abolish production of antibodies which antibodiesreduce or abolish the in vitro secretion of TNF or other cytokine byhuman monocytes or mouse peritoneal macrophages following stimulationwith phospholipid-containing TNF-inducing non-LPS antigens.

The immunogens of the present invention will contain a suitable B-cellepitope. This enables the stimulation of circulating antibodies in anindividual at risk or a host animal and will be sufficient to provideshort term protection for instance for individuals travelling to areaswhere non-LPS organisms are endemic, and will be sufficient to generateantibodies and antibody-secreting cells in host animals which may beused as, or in the production of inhibitors as discussed above.

The immunogens of the invention may also contain an appropriate T-cellepitope in which case they will stimulate memory cells in immunisedindividuals thereby providing long term protection against repeatedexposure to the non-LPS organisms.

Inhibitors as described above may be used as immunogens of the inventionprovided that they are sufficiently large molecules to be immunogenic intheir own right. Inhibitors which are not sufficiently large may bebound to carrier proteins in conventional manner for use as immunogens.Fragments of the TNF-inducing antigens which do not contain thereceptor-binding site as such and fragments of the receptor moleculewhich do not contain the antigen-binding site as such may be used asimmunogens provided that they stimulates production of antibodies whichwill block the antigen: receptor binding. The TNF-inducing antigens, orfragments thereof containing the receptor binding site may be used asimmunogens in accordance with the invention in host animals and may alsobe used to treat humans; where appropriate, at least in the latter case,the TNF-inducing activity will be reduced or abolished, for instance bydegradation as described above. Receptor molecules and fragments thereofcontaining the antigen-binding site may also be used as immunogens ofthe invention in host animals and may also be used to treat humansprovided, in the latter case at least, that any suitable precautions aretaken to avoid stimulating undesirable auto-immunity to the intactreceptor molecules on the patient's cells.

Preferred immunogens of the present invention are PI, PS or PThr lipids,IMP, phosphorylated sugars such as glucose, galactose, fructose andmannose but not glucosamine and degraded TNF-inducing antigens asdescribed above and such materials bound to a carrier protein,preferably tetanus toxoid or keyhole limpet haemocyanin or morepreferably an exoantigen of a non-LPS protozoal parasite.

Phospholipid liposomes and phosphorylated sugars, especiallymonosaccharides may also be used as immunogens according to theinvention is they can generate antibodies against phosphate groups whichcan bind the non-LPS antigens. It is not important which phospholipidsor sugars are used though PI and phosphatidyl serine phospholipidliposomes are preferred.

The immunogens of the invention will be used in conventional manner,preferably in combination with suitable adjuvants such as aluminiumhydroxide gel for humans or Freunds complete or incomplete adjuvant orsaponin for host animals, as ISCOMS or in liposomes. They may beadministered by any conventional route such as intradermally orsubcutaneously, intramuscularly or intravenously and, for animals,intraperitoneally, in the form of suitable pharmaceutical formulations.Preferably the immunogens are presented as solutions or suspensions inwater for injection or as dry powders for reconstitution withpyrogen-free water or water for injection, and may also contain suitablebuffers, antioxidants, preservatives and agents for adjusting thetonicity.

Suitable dosage regimes depend on the desired immune response buttypically involve at least one and possibly several repeat injections atintervals of a few days, such as one or two weeks, up to a few months,such as 1, 2, 3 or 6 months and possibly with boosters at intervals ofone or more years for instance up to 5 years.

Suitable dosages will depend upon the immunogenicity of the materialadministered but will typically be in the range of 1 to 1000 mg for anadult human.

The invention will be further illustrated by the following Example whichis not intended to limit the scope of the invention in any way:

EXAMPLE 1

It has previously been shown that malaria parasites liberate exoantigenswhich, through a phospholipid component, stimulate mouse macrophages tosecrete tumour necrosis factor (TNF) and are toxic toD-galactosamine-sensitised mice, and which therefore might be involvedin pathology. Plasmodium yoelii expamtoges detoxified bydephosphorylation or digestion with lipases do not induce TNFproduction. However, these partial structures inhibited its productionin response to the exoantigens, though not to bacteriallipopolysaccharide (LPS).

When pure phospholipids were tested in a macrophage assay, nonestimulated the production of TNF but phosphatidyl inositol (PI)inhibited TNF induction by P. yoelii exoantigens. Moreover, inositolmonophosphate (IMP) was the only one of a number of monophosphatesaccharides tested which was inhibitory:inositol was not. Macrophagespretreated with PI, IMP or detoxified exoantigens and then incubatedwith parasite exoantigens also yielded much less TNF. PI, IMP andlipase-digested. exoantigens of P. yoelii inhibited the TNF-inducingactivity of exoantigens of the human parasites P. falciparum and P.vivax similarly. Neither PI nor IMP diminished TNF production inresponse to LPS, in contrast to a platelet-activating factor antagonist(1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine)which inhibited both exoantigen and LPS-induced production of TNF.

It is concluded that at least two different parts of the molecule areinvolved in the induction of TNF secretion by parasite exoantigens: onerequires the presence of a phosphate bound to inositol and, sincedephosphorylated exoantigens were also inhibitory, one does not. Itwould seem that both affect interactions between parasite-derivedexoantigens and the machrophage receptors.

INTRODUCTION

Cytokines such as tumour necrosis factor (TNF) and interleukin-1 (IL-1)have been shown to induce fever (15), and the results of a recentclinical trial of a monoclonal antibody against TNF suggest that thefever of malaria is indeed mediated by TNF(29). Furthermore, severalstudies have shown that there is a significant association betweencirculating levels of TNF and the complications of Plasmodium falciparuminfection (19,24,28). The involvement of TNF in the illness andpathology of malaria has been well reviewed by Clark and hisco-workers(14).

It has already been shown that human and rodent malaria parasitesrelease exoantigens, which stimulate macrophages to secrete TNF in vitro(7,41) and are toxic to mice pretreated with D-galactosamine tosensitise them to TNF (8). It would seem that the antigens are releasedat schizont rupture (27) and this could explain the well-knownassociation between fever and this stage of the parasite's developmentalcycle. The exoantigens are highly immunogenic, giving rise to antibodywhich blocks their ability to induce TNF in vitro and protectsD-galactosamine-sensitised mice from their toxic effects (5). We haveproposed that the presence of such antibody might account for the"antitoxic" immunity that is acquired by people living in areas wheremalaria is endemic and have suggested that the exoantigens might becandidates for an antidisease vaccine (34).

Some years ago Clark pointed out (13) that many of the symptoms ofmalaria have features in common with those of endotoxaemia, and it isnow recognised that the latter is associated with TNF induced in thehost by lipopolysaccharide (LPS) released from the bacteria responsiblefor Gram-negative sepsis (11). Indeed, monoclonal antibodies against TNF(43) and against lipid A, the part of the LPS molecule that mediatesmany of the biological activities of LPS including its toxicity, canprotect animals against lethal bacteriaemia (35). In order tocharacterise the molecular structures of LPS that lead to the inductionof septic shock, the activity of various partial structures of LPS havebeen studied. Lipid X, for example, is a non-toxic intermediate in thebiosynthesis of lipid A whose structure resembles half that of the lipidA molecule. Purified lipid X does not induce the production of TNF (2,26, 30, 31) but inhibits various biological activities of LPS in vitroand its toxicity for mice (30, 36). Lipid IV_(A), another partialstructure, similarly failed to induce the production of TNF, but alsoinhibited its induction by LPS; these results have been explained bycompetition for LPS receptors on target cells (26).

Although the phospholipid-containing malaria exoantigens are clearlydifferent from LPS (40) and apparently stimulate macrophages throughdifferent receptors (41), there are some functional analogies. In thepresent Example, various "detoxified" preparations of parasiteexoantigens (that is, those which were not toxic to mice and did notinduce TNF production by macrophages) are tested to see if they couldinhibit its induction by active preparations of parasite exoantigens. Wealso examined the ability of some molecularly defined phospholipids andof some monophosphate saccharides both to induce the production of TNFand to inhibit the TNF-inducing activity of the exoantigens. The resultsobtained with exoantigens derived from the rodent parasite P. yoeliiwere confirmed with samples from the human parasites P. falciparum andP. vivax. The TNF-inducing component of the exoantigens appears to bephospholipid, in that its activity is destroyed not only by digestionwith lipases but more specifically by digestion with phospholipase C; itis also inactivated by dephosphorylation by hydrofluoric acid. Toinvestigate the specificity of this inhibition, we compared the effectsof the various compounds found to inhibit the induction of TNF by theexoantigens for their effects on induction by LPS. Finally, we examinedthe ability of an antagonist of platelet-activating factor (PAF) toinhibit induction by both LPS and the exoantigens. PAF, itself aphospholipid, is an endogenous mediator that causes shock (21) and isreleased in endotoxaemia (12) and it has been shown that a PAFantagonist can block necrosis induced by LPS through the release of TNF(38).

Materials and Methods

Mice. Outbred females at least 6 weeks old were used (Tuck No1; A. Tuck& Sons, Battlesbridge, Essex).

Rodent parasites. The YM lethal variant of P. yoelii (obtained from D.Walliker, Edinburgh University) was used (18). Mice were injectedintravenously with 10¹ parasitized erythrocytes, and parasitaemia wasdetermined from blood films stained with Giemsa.

Preparation of exoantigens. Since TNF-inducing activity was notassociated with protein but was enhanced by its removal, all exoantigenpreparations were incubated for 24 h at 37° C. in 10 μg per ml ofpronase E (Sigma), boiled and dialysed against PBS. No protein was thendetectable by BioRad assay (<1 μg per ml). Before use, they were mixedwith polymyxin B-agarose (Sigma) to remove any endotoxin, centrifuged at500×g for 10 min and sterilised by filtration through a 0.2 μm-pore-sizemembrane filter (Flow Laboratories, Irvine, Ayrshire, United Kingdom)and stored a 4° C.

Exoantigens of P. yoelii. As described previously (39), exoantigens wereprepared from mice with high parasitaemia bled by cardiac puncture. Theerythrocytes were washed twice in sterile phosphate-buffered saline(PBS) and then suspended in PBS at 10^(k) parasitized cells per ml insuspension on a roller at 37° C. for 24 h. Next day the suspensions werecentrifuged at 500×g for 10 min. The supernatant was then boiled for 5min and centrifuged at 1,300×g for 10 min; these supernatants were thenpassed through a membrane filter and stored at 4° C. Detoxification ofP. yoelii exoantigens. (1) By treatment with lipase: Exoantigenpreparations in PBS were incubated overnight at 37° C. with 2-5 U per mlof wheat germ lipase bound to agarose (Sigma) which was the removed bycentrifugation at 500×g for 10 min. The supernatants were sterilised byfiltration and stored at 40° C.

(2) By dephosphorylation: Lyophilised samples were dissolved in 46% HFand kept at 0° C. in polythene tubes for 22 h; they were then dilutedwith PBS and neutralised with NaOH. Precipitates were removed bycentrifugation at 1,300×g for 10 min and the supernatants weresterilised by filtration and stored at 4° C.

Exoantigens of P. falciparum: These were kindly provided by Dr D.Kwiatkowski of Oxford University. They had been prepared by incubatingschizont-enriched preparations from a continuous culture system inminimum essential medium without serum at 37° C. for 24 h. The cultureswere then centrifuged and the supernatants collected and boiled for 5min. The preparations were centrifuged again and the supernatantsobtained were passed through a membrane filter and stored at 4° C.

Exoantigens of P. vivax: These were kindly provided by Prof. K. Mendis,University of Colombo. Erythrocytes obtained from 3 patients infectedwith P. vivax at a time when the parasites had become schizonts werewashed and concentrated to about 80% parasitaemia. They were suspendedin PBS without serum at 1×10.sup. / infected erythrocytes/ml andincubated at 37° C. for 24 h on rollers. Supernatants were collected,passed through a 0.2 μm Millipore filter, boiled for 5 min, centrifugedagain and the supernatants obtained were pooled and stored at 4° C.

Stimulation assays using mouse peritoneal cells. As described previously(5), cells were collected from mice given thioglycolateintraperitoneally 3-5 days previously, using Hanks balanced saltsolution (Flow Laboratories) containing 1 U of heparin and 5 mg ofpolymyxin B (Sigma) per ml. Washed cells were suspended in 5% fetal calfserum in RPMI 1640 (Flow Laboratories) containing polymyxin B andadjusted to 10⁷ viable cells per ml; 0.1 ml volumes were then dispensedinto wells of 96-well microtitre plates (Nunc, Roskilde, Denmark). Thecells were incubated for 2 to 3 h at 37° C. to allow macrophages toadhere and then for 30 min with an equal volume of medium containing 2μg of indomethacin (Sigma) per ml. Non-adherent cells were removed, themedium was replaced by 0.2 ml volumes of RPMI 1640 containing polymyxinB and the stimulants to be tested, and the cultures were incubatedovernight. (Serial dilutions of the stimulants were always tested toestablish a dose-dependent relationship, and experiments were repeatedat least twice). The next day, supernatants were collected and assayedfor TNF by their cytotoxicity for L929 cells. A 1/10 dilution of eachwas made in medium containing 5% fetal calf serum and 1 μg of emetine(Sigma) per ml and stored at -20° C. in case a titration needed to berepeated. Cultures incubated with serial dilutions of LPS or with mediumalone were included in every experiment as positive and negativecontrols on the capacity of the macrophages to yield TNF. Inhibitionassays. The agents to be tested were titrated for their ability to blockthe induction of TNF by mixing equal volumes of serial dilutions with asingle concentration of an exoantigen preparation or of LPS beforeaddition to the acrophage cultures. As phospholipids arelight-sensitive, the plates were covered with aluminium foil.

TNF assays. TNF was assayed colorimetrically by its cytotoxicity forL929 cells (obtained from the European Collection of Animal CellCultures, Porton Down, Salisbury, Wiltshire) seeded at 2.5×10.sup. /cells per well the day before, as described previously (8). Serialdilutions of macrophage supernatants were tested in duplicate in 0.1 mlvolumes per well of 96-well microtitre plates in RPMI 1640 containing 1μg emetine per ml. One unit is defined as the amount causing 50% celldestruction.

Other Reagents. LPS (phenol extract of Escherichia coli (055:B5), allphospholipids (phosphatidyl inositol from bovine liver (PI),phosphatidyl choline from egg yolk (PC), phosphatidyl serine from bovinebrain (PS), phosphatidyl ethanolamine from bovine brain (PE),phosphatidic acid (PA) and cardiolipin from bovine heart (CL) and allthe monophosphate saccharides tested were obtained from Sigma. A PAFantagonist,1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho-(N,N,N-trimethyl)hexanolamine,was obtained from Novabiochem.

Results

Inhibitory activity of detoxified exoantigens. The toxicity of parasiteexoantigens and their ability to trigger macrophages to secrete TNF invitro is mediated by a phospholipid, which accumulates in thechloroform/methanol phase of a two phase lipid extraction process (41),is destroyed by de-O-acylation by mild alkaline treatment and bytreatment with lipases, and also by dephosphorylation and digestion byphospholipase C. We therefore looked to see whether exoantigens of P.yoelii that had been detoxified, either by dephosphorylation or byremoval of fatty acids by lipase digestion, contained structures thatcould inhibit the ability of active preparations to stimulatemacrophages to secrete TNF. Yields of TNF were compared with controlsdiluted in medium. Preparations that had been detoxified by eitherprocedure caused a dose-dependent decrease in the amount of TNFproduced. FIG. 1a shows the results of a typical experiment in which adephosphorylated preparation was included with an active preparationwhich stimulate the production of 102,400 U per ml of TNF; FIG. 1b showsresults (mean±SD) of experiments done with one lipase-digestedpreparation titrated against 3 different active preparations.Lipase-digested supernatants from uninfected mouse erythrocytes were notinhibitory. (As the results shown were obtained both with differentdetoxified exoantigen preparations and with stimulatory preparations,direct comparison of the inhibitory activity of preparations detoxifiedby the two methods cannot be made).

                  TABLE 1                                                         ______________________________________                                        TNF induction by P. Yoelii exoantigens and LPS: Specificity                   of inhibition.                                                                                       Control.sup.a                                                                            Percent                                                            TNF titre  inhibition                                  Inhibitor    Stimulus  (log 4)    (Mean ± SD)                              ______________________________________                                        Lipase-digested                                                                            Exoantigen                                                                              4.9.sup.b  99.7 ± 0.6                               exoantigen                                                                                 LPS       4.2.sup.c  0                                           Dephosphorylated                                                                           Exoantigen                                                                              4.4.sup.d  99.6 ± 0.01                              exoantigen                                                                                 LPS       4.7.sup.e  5.2 ± 7.1                                Lipase-digested                                                                            LPS                                                              10 μg/ml  Exoantigen                                                                              5.5        0                                           1 μg/ml   Exoantigen                                                                              1.8        0                                           1 μg/ml   LPS       3.0        100                                         1 μg/ml   LPS       2.7        93.2                                        0.25 μg/ml                                                                              LPS       2.7        87.4                                        0.06 μg.ml                                                                              LPS       2.7        42.2                                        0.015 μg/ml                                                                             LPS       2.7        24                                          ______________________________________                                         LPS as a stimulus was used at 0.1 μg per ml throughout                     .sup.a in the absence of inhibitor                                            .sup.b mean of 13 experiments                                                 .sup.c mean of 2 experirments                                                 .sup.d mean of 8 experiments                                                  .sup.e mean of 4 experiments                                             

This inhibition of TNF induction was specific to the parasiteexoantigens. These usually induce amounts of TNF of the same order asthose obtained with ca. 1 μg per ml of LPS (41) but none of theexoantigen preparations detoxified by either means inhibited theinduction of TNF by 0.1 μg per ml of LPS (Table 1).

Conversely, detoxified preparations of LPS which inhibited the inductionof TNF by LPS did not affect induction by P. yoelii exoantigens. Forexample, digestion of 10 μg per ml of LPS by lipase totally abolishedits activity and although 1 μg per ml of such detoxified preparationsinhibited the induction of TNF by untreated LPS, 10 μg per ml did notaffect the amount of TNF produced in response to the parasiteexoantigens and 1 μg per ml did not decrease the activity of a dilutedsample.

Inhibition by Phospholipids. As these results indicated that theactivity of the P. yoelii phospholipid component could be inhibitedcompetitively by the inactivated structure, we tested a number ofdifferent commercially available phospholipids to see if they too wereinhibitory. PI consistently inhibited the TNF-inducing activity of theexoantigens; PC, PS, PE, PA and CL, at the same doses, did not (Table2).

                  TABLE 2                                                         ______________________________________                                        Titres of TNF induced by P yoelii exoantigens in                              the presence of various exogenous phospholipids.                                                μg per ml                                                Phospholipid tested     20        4   0.8                                     ______________________________________                                        Expt 1                                                                        None              3.3                                                         Phosphatidyl inositol   0         0   0.8                                     Phosphatidic acid.sup.a 3.2       3.0 3.1                                     Phosphatidyl choline    3.4       3.4 3.4                                     Phosphatidyl serine     3.3       3.4 3.0                                     Phosphatidyl ethanolamine.sup.a                                                                       3.5       3.2 3.3                                     Cardiolipin             3.1       3.4 3.3                                     Expt 2                                                                        None              3.6                                                         Phosphatidyl inositol.sup.a                                                                           0         0   0.4                                     Phosphatidyl choline.sup.a                                                                            3.6       3.6 3.6                                     Phosphatidyl serine.sup.a                                                                             3.6       3.6 3.6                                     Expt 3                                                                        None              4.6                                                         Phosphatidyl inositol.sup.a                                                                           0         0   1.6                                     Phosphatidyl serine.sup.a                                                                             4.6       4.6 4.6                                     ______________________________________                                         Titres of TNF are log 4 after an initial 1/10 dilution of macrophage          supernatants .sup.a free salt.                                           

None of these phospholipids, even at concentrations of up to 100 μg perml, stimulated the macrophages to secrete TNF.

Structural requirements of exoantigen inhibitors. To investigate theminimum structure required to inhibit the stimulation of TNF by the P.yoelii exoantigens, macrophage cultures were incubated with apreparation which induced the secretion of 12, 8000 U TNF per ml andvarious concentrations of either PI, its derivatives inositolmonophosphate (IMP) or inositol. Both PI and IMP caused a dose dependentinhibition of TNF production; the results of representative experimentsare shown (FIG. 2). Inositol itself did not affect the yield of TNF,indicating that the presence of a phosphate bound to the inositol ringis essential for the exoantigen molecule to interact with themacrophage. This was confirmed by tests with other monophosphatesaccharides. Thus, even at 200 μg per ml, the maximum concentrationtested, no inhibition was obtained with glucose-1-phosphate,galactose-1-phosphate, mannose-1-phosphate, fructose-1-phosphate,galactosamine-1-phosphate, glucosamine-1-phosphate, or even adenosinetriphosphate (ATP) (data not shown). Serological cross-reactions areknown to occur between phosphate-containing compounds such as thevarious phospholipids, lipid A, denatured DNA and lipoteichoic acid (1).Our results indicate, however, that unlike antibody recognition ofphosphate esters, recognition by the macrophage receptor of the activemoiety of the parasite exoantigens is highly specific, since itpossesses a site which can recognise phosphate when bound to inositolbut not to other sugars. When an exoantigen preparation was titrated inthe presence or absence of 20 μg per ml of IMP, the yield of TNF wasreduced by about 16-fold at all dilutions and the inhibition was notovercome at the higher concentrations of the stimulant which appeared toreach a plateau, suggesting that the binding of the inhibitor to thisreceptor might not be reversible (FIG. 3). As before, inhibition ofexoantigen activity was specific, in that neither PI nor IMP, even atconcentrations of 20 μg per ml, inhibited TNF induction by LPS (FIG. 4).These results, and our findings of the lack of any effect of thedetoxified exoantigens on the yields of TNF stimulated by LPS, alsoexcluded the possibility that any inhibition observed was due to a toxiceffect of the inhibitors on the macrophages.

Inhibition of exoantigens of other Plasmodium spp. Since TNF-inducingexoantigens of the rodent parasite P. yoelii and of the human parasitesP. falciparum and P. vivax cross-react serologically (5,6), experimentswere done to see if preparations which inhibited the activity ofexoantigens of P. yoelii were also active against those of the humanparasites. The amounts of TNF induced by exoantigens of both P.falciparum and P. vivax decreased in a dose-dependent manner in thepresence of partial structures of P. yoelii exoantigens, in this case,produced by lipase digestion (FIG. 5). Furthermore, incubation with PIand IMP also inhibited the induction of TNF by exoantigens of P.falciparum and P. vivax (FIG. 6).

TNF production by macrophages pretreated with inhibitors. The inhibitoryeffects described above were all observed in experiments in whichmacrophages were incubated overnight in the presence of both stimulantand inhibitor. To distinguish a possible effect of the inhibitor on theexoantigens themselves from an effect on the cells and to investigatethe possibility that the inhibitors acted by blocking cell receptors,macrophages were pretreated with various inhibitors for 30 mins, washed,and then exposed to the exoantigens overnight as usual. Since only smallinhibitory effects were detected in pilot experiments, perhaps due tothe expression of new receptors, exoantigens were removed from thepretreated macrophages after 1 hr in subsequent experiments and thecells were washed before overnight incubation in fresh medium.

The yields of TNF obtained from macrophages that had been pretreatedwith a 1/5 dilution of detoxified P. yoelii supernatants, PI or IMP(both at 20 μg per ml) and then incubated with P. yoelii exoantigenswere greatly decreased, whereas control cultures pretreated with 50 μgper ml of PE or PC were unaffected (FIG. 7. Again, in contrast toresults with exoantigens, pretreatment of the cells with lipase-digestedexoantigens did not diminish the amounts of TNF produced in response toLPS (1 μg per ml) (not shown).

Platele Activating Factor inhibitor. Since the PAF antagonist SRI 63-119has been shown to block a TNF-mediated effect of LPS (38), we examinedthe effects of the antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N-trimethyl)hexanolamine, on TNF productioninduced by P. yoelii exoantigens compared with LPS. No distinction wasfound, as the antagonist caused a dose-dependent inhibition in theyields of TNF obtained in response to both stimulants (FIG. 8).Furthermore, no significant inhibition occurred in response to eitherwhen they were incubated with washed macrophages that has beenpretreated with 50 μg/ml of inhibitor. These findings also showed thatthe antagonist did not have a toxic effect on the macrophages.

DISCUSSION

Our most striking finding is that the TNF-inducing activity of moleculesreleased into the medium by erythrocytes infected with P. yoelii, orwith the human parasites P. falciparum and P. vivax can be inhibited,clearly and reproducibly, by the simple, chemically defined moleculesphosphatidyl inositol and inositol monophosphate and by parasiteexoantigens modified to render them non-toxic. These results support ourprevious conclusion that the active component of the exoantigens of P.yoelii depends upon a phospholipid and provide more information aboutthe nature of this phospholipid.

Two lines of evidence indicate that the inhibitory activity of PI is notmedicated through its diacylglycerol component: first, diacylglycerol ispresent in all the phospholipids tested that had no inhibitory effectand secondly, exoantigens from which it has been removed by lipasedigestion are still inhibitory. Furthermore, IMP, an unacylated form ofPI, was also inhibitory. Such inhibition occurred when covalently boundphosphate and inositol were present; neither inositol itself nor anumber of other monophosphate saccharides were effective. Thespecificity of this inhibition suggests that an inositol phosphate groupmust be an integral part of the active structure of the exoantigens.

It has been noted that the lipid chemistry of different species ofPlasmodium is remarkably similar and differs considerably from that oferythrocytes (3) and that phospholipids of the parasitophorous vacuole(3) and of the rhoptries of merozoites are discharged at the time ofinvasion (37). The incorporation of inositide into phosphatidyl inositolhas been shown to be accelerated during early schizogony (44).Phosphatidyl inositol is a constituent of several antigens of theparasite, including, for example, a merozoite surface antigen (20),forming an essential part of the glycosyl phosphatidyl inositol (GPI)anchor that attaches some proteins to surface membranes (reviewed in 17and 42). We do not know if the inositol phosphate-containing portion ofthe exoantigen is covalently bound to a particular protein antigen ormerely associates with protein because of its hydrophobic properties butthere is reason to believe that it is not a GPI anchor. Thus, nitrousacid deamination of molecules containing these anchors liberates free PI(17,42) and we have shown both that PI does not induce TNF productionand that the TNF-inducing activity of the exoantigens is unaffected bydeamination. Furthermore, the presence of glucosamine is diagnostic forGPI anchors and they generally contain mannose. Our failure to inhibitthe activity of the exoantigens using either glucosamine-1-phosphate ormannose-1-phosphate (or using glucosamine or mannose, data not given,suggests that these sugars if present are not a necessary part of theantigen ligand; we have also showed (data not given) that sugar moietiesare not necessary for exoantigen induction of TNF. Inositolmonophosphate also inhibits the activity of phosphatidyl inositolglycans (PI-Gs) which mimic the action of insulin on adipocytes (32),probably by competing for receptors. However, in contrast to theparasite exoantigens, glucosamine and mannose also inhibit the activityof the PI-Gs, and deamination by nitrous acid abolishes theinsulin-mimicking effect (17).

The inhibitory effect of PI, IMP and the detoxified exoantigens on theTNF-inducing activity of the exoantigens might be explained in two ways:the inhibitors might pass across cell membranes directly and thusinhibit a second messenger system but our findings indicate that thiswould have to be one which is activated in macrophages stimulated byexoantigens but not by LPS. Alternatively, they may act by competing forspecific receptors on the macrophage. The fact that detoxifiedexoantigens were inhibitory, whether they had been dephosphorylated orde-O-acylated by lipase digestion, suggests that there may be at leasttwo binding sites on the macrophage receptor whose occupancy leads toinhibition: one depending on the presence of phosphorus and one not. Forthe exoantigens to induce the production of TNF, however, ester-linkedacyl chains must also be present (data not shown). We can only speculateas to whether they bind to a third site on the receptor or act on thepathway which leads to the synthesis of TNF by some other route.

Cytokine production by LPS is also abolished by deacylation, andinactive endotoxin derivatives block TNF production (26) and B cellmitogenesis (16) by the active molecule. It is noteworthy that thedetoxified exoantigens, PI and IMP did not block the induction of TNF byLPS, confirming our suggestion (41) that the exoantigens and LPSstimulate macrophages via different receptors. The PAF antagonistinhibited TNF induction by both stimulants when incubated with them, butnot when used to pretreat macrophages before addition of the stimulants,suggesting that PAF is generated as a common second messenger.

Macrophages bear several classes of scavenger receptor which haveaffinities for oxidized and acetylated low density lipoproteins (LDL)and which have a broad binding specificity (10, 25). The binding ofoxidized LDL to mouse macrophages is inhibited by liposomes containingacidic phospholipids such as PE, PS and PI, and it appears that at leasttwo scavenger receptors recognise these molecules (33). This suggestedto us that the inositol phosphate-containing exoantigens might bind tothese receptors. We found that exoantigen-induced TNF secretion wasindeed inhibited by oxidised by not native LDL. Like Hamilton et al.(22), we found that oxidized LDL itself did not induce peritonealmacrophages to secrete TNF, and that it did not inhibit the TNF-inducingactivity of LPS. However, in preliminary experiments we could not detectsignificant inhibition of exoantigen activity by such scavenger receptorligands as fucoidan, dextran sulphate and polyinosinic acid andfurthermore macrophages pretreated with oxidized LDL still responded tostimulation by the exoantigens. While such findings depend greatly uponthe experimental conditions used, the possibility that oxidised LDL wasinhibitory because it formed a complex with the exoantigens should beconsidered. Work with endotoxin has shown that the binding of the LPSderivative lipid IV_(A) to a mouse macrophage cell line is mediated by ascavenger receptor since it is inhibited by acetylated LDL (23). Bindingto this receptor did not stimulate macrophages to secrete TNF, althoughothers have reported that acetylated LDL at very high concentrationsstimulated human monocytes to secrete a short burst of TNF that haddisappeared from the medium by 24 hr (4). Since scavenger receptorligands inhibited hepatic uptake of lipid IV_(A) in mice, it wassuggested that such a receptor may be involved in the clearance anddetoxification of endotoxin in vivo (23).

The toxic properties of the exoantigens may explain some of the clinicalcomplications of malaria and, if so, modified exoantigens might form thebasis of an anti-disease vaccine (34). The findings reported hereprovide another approach to the management of malaria. The toxicity ofinfection might be reduced specifically by administration of exoantigenpartial structures, or by other simpler inhibitors. Thus, in addition tomeasures designed to counteract the harmful effects of cytokines, suchas the administration of monoclonal antibodies or of cytokineinhibitors, this kind of therapy, which should act earlier in thetoxigenic pathway, might prevent the production of excess TNF andperhaps of other cytokines or damaging processes that might be activatedby these parasite exoantigens.

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25. Kodama T., M. Freeman, L. Rohrer, J. Zabrecky, P. Matsudaira and M.Krieger. 1990. Type I macrophage scavenger receptor contains α-helicaland collagen-like coiled coils. Nature (Lond.) 343: 531-535.

26. Kovach N. L., E. Yee, R. S. Munford, C. R. H. Raetz and J. M.Harlan, 1990. Lipid IV_(A) inhibits synthesis and release of tumornecrosis factor induced by lipopolysaccharide in human whole blood exvivo. J. Exp. Med.172: 77-84.

27. Kwiatkowski D., J. G. Cannon., K. R. Manogue, A. Cerami, C. A.Dinarello and B. M. Greenwood. 1989. Tumour necrosis factor productionin Falciparum malaria and its association with schizont rupture. Clin.Exp. Immunol. 77: 361-366.

28. Kwiatkowski D., A. V. S. Hill, I. Sambou, P. Twumasi., J.Castracane, K. R. Manogue, A. Cerami, D. R. Brewster and B. M. Greenwood1990. TNF concentration in fatal cerebral, non-fatal cerebral, anduncomplicated Plasmodium falciparum malaria. Lancet 336: 1201-1204.

29. Kwiatkowski D., M. E. Molyneux, F. Pointaire, N. Curtis, N. Klein,M. Smit, R. Allan, S. Stephens, G. E. Grau, P., Holloway, D. R. Brewsterand B. M. Greenwood. 1992. Monoclonal anti-TNF antibody in the treatmentof childhood cerebral malaria. New Engl. J. Med. Submitted.

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31. Lasfargues A. and Chaby R. 1988. Endotoxin-induced tumor necrosisfactor (TNF): selective triggering of TNF and interleukin-1 productionby distinct glucosemine-derived lipids. Cell. Immunol. 115: 165-178.

32. Machicae, F., J. Mushack, E. Seffer, B. Ermel and H-U. Haring, 1990.Mannose, glucosamine and inositol monophosphate inhibit the effects ofinsulin on lipogenesis. Biochem. J. 266: 909-916.

33. Nishikawa K., H. Arai and K. Inoue. 1990. Scavengerreceptor-mediated uptake and metabolism of lipid vesicles containingacidic phospholipids by mouse peritoneal macrophages. J. Biol. Chem.265: 5226-5231.

34. Playfair, J. H. L., J. Taverne, C. A. W. Bate and J. B. de Souza1990. The malaria vaccine: anti-parasite or anti-disease? Immunol.Today. 11: 25-27.

35. Pollack, M., A. A. Raubitchek and J. W. Larrick. 1987. Humanmonoclonal antibodies that recognise conserved epitopes in thecore-lipid A region of lipopolysaccharides. J. Clin. Invest. 79:1421-1430.

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41. Taverns, J., C. A. W. Bate, D. A. Sarkar, A. Meager, G. A. W. Rookand Playfair, J. H. L. 1990. Human and murine macrophages produce TNF inresponse to soluble antigens of Plasmodium falciparum. Parasite.Immunol. 12: 33-43.

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Example 2 Abstract

Phospholipid-containing antigens of malaria parasites stimulatemacrophages to Secrete tumour necrosis factor (TNF), inducehypoglycaemia and are toxic to mice. This TNF induction is inhibited byantisera made against the antigens, the inhibitory activity of which canbe removed specifically by adsorption to phosphatidylinositol (PI)liposomes. Although the same was true of antisera made against PI, theinhibitory activity of antisera made against some other phospholipidsappeared to be directed against a common determinant, probably thephosphate ester head group. We have shown previously that the activityof all the antisera was associated mainly with IgM and was not boostedby repeated injections of the antigens. To try and induce a secondaryresponse against the parasite antigens using non-toxic molecules, micewere immunized with various phosphorylated compounds coupled to keyholelimpet haemocyanin (KLH). Three injections of PI-KLH or ofphosphatidylserine (PS) coupled to KLH induced significantly highertitres of inhibitory antibody than one; furthermore, the inhibitoryactivity was mainly in the IgG fraction. The antisera does not inhibitTNF induction by LPS or lipoteichoic acid. However antisera againstPS-KLH, though not PI-KLH, inhibited the induction of TNF by thephospholipid, platelet-activating factor (PAF). These antisera, andantisera from mice immunized with phospho-threonine orgalactosamine-1-phosphate conjugated to KLH, contained inhibitoryantibodies of differing specificities. Mice immunized with PI-KLH,PS-KLH or phosphothreonine-KLH did not develop hypoglycaemia whenchallenged with the parasite toxic antigens. These results indicate thatthe antigenicity of non-toxic analogues can be dramatically enhanced bycoupling to a protein carrier.

Introduction

While tumour necrosis factor (TNF) is widely accepted as playing animportant role in septic shock (1), its possible involvement in theillness and pathology of malaria has only recently been recognised, thesubject has been well reviewed by Clark and his co-workers (2). Inpatients infected with Plasmodium falciparum, increased levels ofcirculating TNF are associated with the severity of the disease (3,4),and especially with death from cerebral malaria (5). Among its manyproperties, TNF can cause various changes in vascular endothelium. Forexample, it can increase the expression of adhesion molecules such asICAM-1 (6), one of the molecules that may be concerned in the attachmentof parasitized erythrocytes to the endothelium of brain capillaries (7).It can also increase the production of nitric oxide from endothelialcells (8), which if it occurred in the cerebral capillaries might alterthe activity of underlying neurons (9).

Substances released by both rodent and human malaria parasites stimulatemouse macrophages (10) and human monocytes (11,12) to secrete TNF.Furthermore, those from both rodent parasites and P. falciparum aretoxic, in that they kill mice sensitised to TNF by treatment withD-galactosamine (13), suggesting that they might be involved in thepathology of infection. We have shown that the induction of TNF by theseparasite antigens depends upon a phospholipid component (14) and thatthis activity can be inhibited by the addition of phosphatidylinositol(PI) or inositol monophosphate (IMP), suggesting that the active moietycontains PI (15); furthermore, the inhibitory activity of antibodiesagainst these antigens was specifically adsorbed by liposomes containingPI (16).

We have previously referred to these TNF-inducingphospholipid-containing antigens as "exoantigens", as they are presentin the medium of cultures of P. falciparum and in supernatants fromrodent malarial parasites incubated overnight at 37°. Since they arealso present in intact, as well as lysed, parasitized erythrocytes,(14), it would seem more correct to refer to then as toxic antigens.

Immunization with the toxic antigens of P. yoelii prevents mortality ingroups of mice treated with D-galactosamine, before challenge with theantigens, and protection is associated with antibody that inhibits theantigen-induced TNF secretion in vitro (13). We therefore proposed thatthese antigens might form the basis of an anti-disease vaccine (17). Ourfinding that the TNF-inducing antigens from different rodent parasitesand from P. falciparum and P. Vivax are serologically related (18, 19)suggests that they are conserved. However, since they are not only toxicbut give rise mainly to T-independent antibody which is predominantlyIgM (19), the immunogen of choice would probably need to be renderednon-toxic and modified so as to generate immunological memory.

We had found that a number of compounds, including PI and its derivativeIMP and phosphatidylserine (PS) (in liposomal form), none of whichstimulate TNF secretion by macrophages, also induce the production ofinhibitory antibodies; again these were predominantly IgM and theirtitres were not enhanced repeated injections (16). A memory responsehas, however, been induced to the organophosphorus neurotoxin soman,coupled to keyhole limpet haemocyanin (KLH) (20). Therefore, to try andinduce high titre inhibitory antibody and immunological memory, using anon-toxic phospholipid as an immunogen, we coupled PI, PS and some otherphosphorylated compounds to KLH immunized mice with them andinvestigated their antibody responses.

Hypoglycaemia is known to be associated with severe malaria (21) and wehave shown that supernatants from blood stage P. yoelii inducehypoglycaemia in mice, with a time course different from that seen afterinjection of insulin (22). The antigens which precipitate a fall inblood glucose levels in vivo resemble those which induce the productionof TNF in vitro in a number of ways, suggesting that they too might bePI-containing phospholipids (22). However a monoclonal antibody whichneutralised the activity of murine TNF did not block the hypoglycaemicresponse to the toxic antigens. Since mice immunized with the antigenswere protected against the antigen-induced hypoglycaemia, we have alsoexamined mice immunized with some of the KLH conjugates to see if theytoo were protected.

Materials and Methods

Mice

Antisera were generated in outbred female mice which were at least 6weeks old (Tuck No1: A. Tuck & Sons, Battlesbridge, Essex and MF-1:Harlan Olac, Bicester, Oxon). Hypoglycaemia experiments were done in(C57 B1×Balb/c) F1 females at least 10 weeks old.

Toxic antigens

From P. yoelii YM (obtained from D. Walliker, Edinburgh University,Edinburgh, UK): These were prepared as previously described (14), byincubating washed parasitized erythrocytes, obtained from mice with highparasitaemia, at 10⁸ parasites/ml in PBS overnight on a roller at 37°.Next day supernatants were collected, centrifuged, boiled for 5 min, andcentrifuged again. There were then incubated for 24 h at 37° C. in 10 μgper ml of pronase E (Sigma), since TNF-inducing activity is notassociated with protein but is enhanced by its removal (14). They werethen boiled and dialysed against PBS, when no protein was detectable byBioRad assay (<1 μg per ml). Before use, they were mixed with 5 μg/ml ofpolymyxin B-agarose (Sigma) to remove any endotoxin, centrifuged, passedthrough a 0.2 μm filter and stored at 4° C.

From P falciparum

These were obtained from cultures of P. falciparum maintained in Group Oerythrocytes in RPMI 1640 containing 10% A⁻ human serum. Schizonts wereenriched by plasmagel, washed 3 times, resuspended at 5×10⁻ per ml inserum-free RPMI 1640 and incubated overnight at 37° C. Next day thecultures were vortexed for 30 secs, centrifuged at 10,000×g for 10 minand the supernatant collected and stored at 4° C. for use.

Stimulation assays: (i) using mouse peritoneal macrophages

The cells were prepared as described (14). Briefly, they were collectedfrom mice given 1 ml of 4% thioglycollate (Difco) i.p. 3-5 dayspreviously, using Hank's BSS (Flow Laboratories) containing 1 U/ml ofheparin and 5 μg/ml of polymyxin B (Sigma). Washed cells were suspendedin 5% foetal calf serum (FCS) in RPMI 1640 containing 5 μg/ml polymyxinB, at 1×10⁻ viable cells per ml and 0.1 ml volumes were dispensed intowells of 96-well microtitre plates (Nunc) and incubated for 1-2 hr.

Adherent cells were incubated for 30 min with an equal volume of mediumcontaining 2 μg/ml indomethacin (Sigma) which was then replaced by 0.2ml volumes of serial dilutions of the stimulants to be tested, made inRPMI 1640 containing polymyxin B except in the case oflipopolysaccharide (LPS) controls! and the cultures were incubatedovernight. Next day, supernatants were collected for assay for TNF; theywere stored at -20° in medium containing 5% FCS and 1 μg/ml of emetine(Sigma). Cultures incubated with serial dilutions of LPS (phenol extractof Escherichia Coli 055:B5 Sigma) and with medium alone were included inevery experiment as positive and negative controls for the capacity ofthe macrophages to yield TNF.

(ii) using human peripheral blood mononuclear cells (PBMNC)

Heparinised blood was mixed with an equal volume of saline andmononuclear cells were isolated on Lymphprep (Nyegaard, Oslo, Norway).They were washed twice, resuspended in serum free MEM at 1×10⁵ cells perml, and dispensed in 100 μl volumes in flat bottomed 96 well microtitreplates. 100 μl per well of MEM or antiserum dilutions made in MEM, werethen added, followed by 100 μl of stimulant. The plates were incubatedovernight at 37° C. and the supernatants were then harvested and assayedfor TNF.

Antisera

To compare the inhibitory activity of antiserum to IMP against toxicantigens of both P. falciparum and P. yoelii tested on both humanmonocytes and mouse macrophages, groups of mice were injected i.p. with200 μg of IMP (Sigma), bled 12 days later and their serum pooled. Forthe induction of secondary responses, various phosphorylated compoundswere conjugated to protein by mixing 400 μg/ml in PBS at pH 5.0 with 400μg/ml of KLH (Calbiochem) on ice. They were: PI (from soya bean orbovine liver, Sigma), PS (from bovine brain; Sigma),O-phospho-DL-threonine (Sigma) and D-galactosamiine-1-phosphate (Sigma).They were then mixed with an equal volume of ice cold 150 mM1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC: Sigma), vortexed andleft for 1 hr on ice, when excess EDC was quenched by addition of lysine(Sigma) to 1 mg/ml, and they were then dialysed. Groups of mice wereinjected i.p. at fortnightly intervals with 0.5 ml (which would havecontained 100 μg of the phosphorylated compound if it had all beenconjugated to the KLH) and they were bled 12 days after the lastinjection. Other groups of mice were also immunized similarly with 3injections of 200 μg PI or 0.5 ml of P. falciparum toxic antigens.Antisera were heat-inactivated at 56° C. and before use in the humanPBMNC system they were adsorbed with human erythrocytes. They weretitrated for their ability to block the induction of TNF by the parasitetoxic antigens by mixing equal volumes of serial dilutions made in serumfree medium with one dilution of an antigen preparation, chosen so thatit was on the linear portion of the dose response curve, before additionto macrophages. It was found to be important to perform titrations usingcells that had been washed in serum free medium. Titres are defined asthe reciprocal of the dilution that reduced the amount of TNF producedby 50 percent. (Direct comparisons cannot be made between the inhibitorytitres of the antisera against the different compounds, however, as theefficiency of conjunction was not determined, so that differencesbetween the magnitude of responses might reflect concentration ofimmunogen rather than immunogenicity).

Determination of Ig isotopes

Antisera were depleted of IgG or IgM on isotope-specific agarose (Sigma)and the antibody was eluted with 0.1M glycine HCl at pH 2.5 into 1.0MTris buffer at pH 8, dialysed against PBS and returned to the originalvolume.

Adsorption by liposomes

Multilamellar dehydration-rehydration vesicles (16) were kindly suppliedby Prof. G. Gregoriadis (School of Pharmacy, London University).Antisera diluted 1/50 in RPMI were incubated for 1 hr at roomtemperature with 2 mg/ml of the liposomes, which were then deposited bycentrifugation; this was repeated twice.

Cytotoxicity assay for murine TNF

Samples were assayed colorimetrically by their cytoxicity for L929 cellsas described previously (14). Serial dilutions were tested in duplicate,in 0.1 ml volumes/well, in RPMI containing 1 μg/ml emetine. One unit isdefined as that causing 50% cell destruction.

ELISA assay for human TNF

Microtitre plates (Nunc Immunoplate Maxisorp) were coated overnight at37° C. with 5 μl/well of carbonate buffer pH 9.0 containing 5 μg/ml of amonoclonal antibody against human TNF (CB0006; a kind gift fromCelltech, UK). The plates were washed in PBS containing 0.02% Tween 20and then incubated for 2 hr at 37° C. with 50 μl/well of the samples tobe tested. They were then washed again and incubated for 1 hr at 37° C.with 50 μl/well of a rabbit antiserum against human TNF (Endogen)diluted to 1 μg/ml in PBS/Tween containing 2% normal goat serum. Theywere then washed and incubated for 1 hr at 37° with goat anti-rabbit Igconjugated to alkaline phosphatase (Sigma), when they were washed andthe colour developed with p-nitrophenylphosphate tablets a 1 mg/ml(Sigma) in diethanolamine buffer pH10.5 and the OD 405 nm read on aDynatech Microelisa Autoreader.

Measurement of blood glucose

Glucose concentrations were determined from a drop of tail blood fromthe mice, using Glucostix and an Ames Glucometer (Miles Lid, StokePoges, Slough, UK) according to the manufacturer's instructions (22).Results are expressed as means (±SD) of groups of at least 3 mice.

Other Reagents

Lpoteichoic acid from Staph. aureus was obtained from Sigma and plateletactivating factor (PAF) from Calbiochem.

Results

Mouse macrophages and human monocytes secrete TNF in response to toxicmalaria antigens both rodent and human parasites (11) and this TNFproduction can be inhibited by antisera against the toxic antigens fromeither source, since they cross-react (18,19) as well as by antiseramade against PI and IMP (16). When we compared the inhibitory activityof 5 batches of antiserum against IMP, titrated numerous times usingpreparations of either P. falciparum or P. yoelii in human or mousecells respectively, we found that the inhibitory titres were notsignificantly different (FIG. 9). Therefore, in the work described belowwe have pooled results obtained from the two systems.

Inhibitory activity of antiserum against PI conjugated to KLH

Antisera obtained after 3 injections of PI-KLH were about 100 times moreeffective at blocking the induction of TNF by the toxic antigens thanthose obtained after one injection (FIG. 10a). In contrast to ourfindings with antisera against the toxic antigens of P. yoelii, P.falciparum and P. vivax (18,19) and with antisera against IMP (16) andunconjugated PI (unpublished work), in which the activity waspredominantly associated with IgM most of the activity was now found toreside in the IgG fraction, although IgM antibodies were stilldetectable (FIG. 10b).

We had previously found that there are at least two kinds of inhibitoryantibody, one generated in response to the parasite toxic antigens, orto PI or IMP, which specifically recognises PI in liposomal form, andanother which is less specific and seems to recognise a broad range ofphospholipids (16). To investigate the specificity of the antibodypresent after 2 injections of PI-KLH we adsorbed the antiserum withvarious kinds of liposome (FIG. 10c). The inhibitory activity was notadsorbed by PC, was reduced by PS, and was completely lost afterincubation with PI liposomes.

Inhibitory activity of antiserum against conjugated to KLH

The finding that the inhibitory activity of antiserum against PI-KLH waspartially removed by adsorption with liposomes containing PS suggestedthat inhibitory antibodies exist which bind to acidic phospholipids butnot to the neutral phospholipid PC. We therefore examined the ability ofantisera raised against PS-KLH to inhibit the induction of TNF by thetoxic antigens. Again titres were greatly enhanced after 3 injectionscompared with those of antiserum obtained after one injection (FIG.11a). This activity was remarkable, in that we had previously found thatin contrast to PI, immunisation with unconjugated PS did not induce theproduction of inhibitory antiserum unless it was in liposomal form (16).Again most of the activity was in the IgG, rather than the IgM fraction(FIG. 11b). However, the antisera showed a different specificity fromthose against PI-KLH in that the inhibitory activity was largely removedby adsorption with PS, but was not significantly affected by PI,liposomes (11c). Pretreatment of macrophages with the antisera did notaffect their subsequent ability to respond to the parasite antigens,including that inhibition was not due to any binding of antibody toreceptors on the cells. We conclude that PS-KLH stimulates a differentpopulation of B cells from PI-KLH, hut both populations secreteantibodies which block the TNF-stimulating activity of the parasiteantigens.

Prolongation of antibody production

We had previously found that inhibitory antibody stimulated by oneinjection of P. yoelii toxic antigens was no longer detectable 30 dayslater (19). To see if its production was prolonged by immunization witha conjugated phospholipid, groups of mice were bold at intervals up to 2months after the third injection of PI-KLH or PS-KLH and their seraassayed for inhibitory activity and compared with the results oftitrations of antisera obtained after 3 injections of unconjugated PI orP. falciparum antigens (FIG. 12). Activity as great as the peak activityof serum from mice given the toxic antigens or unconjugated PI waspresent within 3 days. Furthermore its activity was strikingly prolongedand was still present 60 days later. At its peak the titre was more than10 times higher.

Antisera against other phosphorylated compounds conjugated to KLH

We previously found that a number of other phosphorylated compounds alsostimulated the production of inhibitory antibody (unpublished work). Todetermine the effect of conjunction to KLH on their ability to inducethe production of inhibitory antibody, mice were immunised with two ofthem selected for conjunction because they contained amino groups,namely phospho-threonine (P-Thr) and galactosamine-1-phosphate(Gal-1-P), and the inhibitory activity of their serum was compared withthat of mice immunised with PI-KLH and PS-KLH (FIG. 13). No activity wasdetected in serum from a group of mice injected with lysine-KLH,indicating that the inhibitory antibody was induced by thephosphorylated molecules and not by the KLH carrier (or even by theEDC). The titres of Gal-1-P-KLH antisera were similar to those obtainedwith PI-KLH and PS-KLH, while those of P-Thr-KLH antisera were about10-fold less. Again, titres were significantly higher after 3 injectionsthan after 1 (data not shown). However the specificities of the antiseradiffered: thus the inhibitory activity of P-Thr-KLH antisera was removedby adsorption to PC or to PI liposomes (FIG. 14a), whereas PC liposomeshad no effect on antisera against Gal-1-P-KLH, PI liposomes removed someof the activity, and PS liposomes removed most (FIG. 14b). It seems thatantibodies against P-Thr-KLH bind to a comment epitope shared by manyphospholipids, probably the phosphate ester head group. By contrast, thefact that inhibitory antibodies against Gal-1-P-KLH were adsorbed byliposomes containing the two acidic phospholipids, PS and PI, suggeststhat charge might be a factor that affects their binding (23).

TNF secretion induced by other stimulants

Since phospholipids are essential components of every cell membrane, itwas possible that these antiphospholipid antibodies might bind to hostcells. However the inhibitory activity of our antisera was notdiminished by adsorption to normal mouse or human erythrocytes. Sinceantibodies against Plasmodium toxic antigens do not inhibit TNFinduction by LPS (19), and antibodies against PI,IMP and variousphospholipid liposomes which block TNF induction by the parasiteantigens similarly are not active against LPS (16), we incubated PBMNCwith some other stimulants known to induce the production of TNF, namelylipoteichoic acid (24) and platelet activating factor (PAF) (25), in thepresence and absence of a 1/1500 dilution of antiserum against PI-KLH orPS-KLH (FIG. 15). At this dilution both antisera significantly reducedthe amount of TNF produced in response to P. yoelii and P. falciparumantigens. They did not diminish the yield in response to LPS orlipoteichoic acid. However, while PI-KLH antiserum also did not affectthe response to PAF, PS-KLH antiserum significantly inhibited theability of this phospholipid to induce PBMNC to secrete TNF.

Induction of hypoglycaemia in immunized mice

Mice immunized with P. yoelii toxic antigens do not show the same dropin blood glucose 4 hr after challenge with the toxic antigens as docontrol mice (22). We therefore challenged mice that had been immunizedwith some of the KLH conjugates similarly and measured their bloodglucose at various times to see if they too were protected (FIG. 16).While the levels of unimmunized mice and of a group of mice that hadbeen immunized with lysine-KLH were significantly lower at 2, 4 and 8hr, those of mice immunized with PI-KLH did not drop below normal at anytime; although those of mice immunized with PS-KLH and P-Thr-KLH showeda slight drop at 4 hr, they remained significantly higher than those ofthe control groups at all times.

Discussion

Three injections of the phosphorylated compounds PI, PS and Gal-1-Pconjugated to KLH induced the production of high titres of antibodiesthat inhibited TNF induction by toxic malaria antigens. These appearedto be mainly IgG and more long lived than those made against theparasite phospholipid antigens, which were mainly IgM. The mean titreobtained after 3 injections of PI-KLH, for example, was 2-300,000compared with the titres of 8-10,000 elicited by repeated injections ofunconjugated PI or of the parasite antigens. There is evidence that theparasite antigens are normally associated with protein, in that theirability to induce the production of TNF is increased by treatment with aprotease (14). Thus it might be expected that they would be capable ofinducing a secondary IgG response without conjunction to a carrier, butwe have found this not be be so (19).

The immunogens used were selected for a number of reasons: PI because itappears to form a critical part of the TNF-triggering parasite molecule(15,16); PS because it is also a negatively charged phospholipid; P-Thrbecause like PS and Gal-1-P it contains an amino group to which KLHcould be coupled; Gal-1-P because it was one of a number ofphosphorylated sugars, including mannose, glucose, galactose andfructose (but not glucosamine, oddly enough) which induced inhibitoryantibody after 1 injection of 200 μg (unpublished work).

Of the four compounds, P-Thr-KLH gave rise to the lowest titres,although these were significantly higher than those obtained afterinjection of unconjugated P-Thr (unpublished work) and to the leastspecific antibodies. PS-KLH gave rise to the most specific, judging bythe results of adsorption experiments with the different sorts ofliposomes. As we argued previously, since PI inhibits TNF induction bythe parasite antigens (15), and the inhibitory activity of antisera madeboth against the parasite antigens and against inositol monophosphatewas removed specifically by PI liposomes (16), the active portion of theantigens seems likely to contain PI. However antibodies against PI-KLHwere less specific than those obtained against unconjugated PI (16),since they also adsorbed to PS liposomes. This may arise because thebinding of some inhibitory antibodies may be influenced by charge;liposomes incorporating PI and PS express a negatively charged surface,in contrast to those containing PC or cardiolipin, which are neutral(23). Although charge might also explain the specificity of the antiseraagainst PS, it is possible that PS also forms part of the active moiety.

In the light of our previous findings (19,16), it was not surprisingthat these antisera did not inhibit TNF induction by LPS. Furthermore,they were also inactive against lipoteichoic acid, a component of thecell walls of Gram positive bacteria that containspoly(glycerophosphate) chains. Whereas in cur experiments Lipoteichoicacid from Staph. aureus induced the secretion of TNF from human PBMNC,others have reported that it was inactive (24); however, theintraspecies variation they observed may account for our differences.PAF has been reported to stimulate human monocytes to secrete TNF invitro (25). The PAF antagonist,1-0-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, inhibits the induction of TNF by both LPSand by the toxic antigens of P. yoelii, implying that PAF might begenerated as a second messenger in both cases (15). It was surprisingthat antiserum against PS-KLH but not against PI-KLH inhibitedPAF-induced TNF secretion by PBMNC. As macrophages stimulated withcytokines are known to release PAF into the culture supernatant (26), itis possible that it reacted with extracellular PAF, however thisexplanation is unlikely since this antiserum did not also inhabitLPS-induced TNF secretion. Furthermore, although PAF is a modified PC,antibodies against PS-KLH were not removed by adsorption with PCliposomes. It is possible that different epitopes exist on PAF (27)which might not be expressed on PC liposomes or that inhibitoryantibodies that might react with PC do not react with it in liposomalform.

Some anti-phospholipid antibodies cross-react with DNA (28) and theelicitation of such antibodies by parasite toxic antigens releasedduring infection might explain why DNA-binding antibodies are found inthe serum of patients with malaria (29). DNA-binding antibodies are alsoassociated with autoimmune diseases such as systemic lupuserythematosis, raising the possibility that immunization withphospholipids might induce harmful autoimmune reactions. However, nopathological consequences have been reported in phase I and II trials ofdrug-containing liposomes (30) and experimental animals given repeatedinjections of phospholipids do not develop autoimmune disease.Furthermore, differences have recently been shown to exist betweenantiphospholipid antibodies associated with autoimmune disease and thoseassociated with infection, including malaria (31).

We have observed that our toxic parasite antigens cause hypoglycaemia aswell as induce TNF secretion. This does not appear to be due to theability of TNF itself to induce hypoglycaemia (32) as pretreatment ofmice with a monoclonal antibody against TNF did not prevent them fromdeveloping hypoglycaemia when challenged with the parasite toxicantigens (22). The finding that immunisation with PI, PS and P-Thrconjugated to KLH prevented the antigen-induced hypoglycaemia providesfurther support for our view that these hypoglycaemia-inducing moleculesare also phospholipids. We suggested earlier that the toxic antigens ofmalaria, suitably detoxified and modified to induce IgG and memory,might form the basis of an anti-disease vaccine (17), which wouldprotect individuals against the toxicity of malaria, whether mediatedthrough the induction of cytokines or by other mechanisms. The resultsdescribed here indicate one way in which such a goal might be achieved.

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We claim:
 1. A method of treating or preventing clinical manifestationsof malaria, caused by infectious plasmodial organisms which expressantigens which in the patient stimulate secretion of harmful levels ofat least one cytokine, but which do not stimulate secretion of cytokinesonly by expression of lipopolysaccharide, which method comprisesadministering to a human in need thereof an effective non-toxic amountof an immunogen, wherein said immunogen is a pharmacologicallyacceptable material comprising inositol monophosphate or a phosphatidylinositol, which immunogen stimulates production of antibodies which, invitro, reduce or abolish secretion, by at least one of human monocytesand mouse peritoneal macrophages, of tumour necrosis factor followingstimulation with a phospholipid-containing tumour necrosisfactor-inducting antigen other than lipopolysaccharide.
 2. A materialfor use in the treatment or prevention of clinical manifestations ofmalaria, caused by infectious plasmodial organisms which expressantigens which in the patient stimulate secretion of harmful levels ofat least one cytokine, but which do not stimulate secretion of cytokinesonly by expression of lipopolysaccharide, which material is animmunogen, wherein said immunogen is a pharmacologically acceptablematerial comprising inositol monophosphate or a phosphatidyl inositol,which immunogen stimulates production of antibodies which, in vitroreduce or abolish secretion, by at least one human monocytes and mouseperitoneal macrophages, of tumour necrosis factor following stimulationwith a phospholipid-containing, tumour necrosis factor-inducing antigenother than lipopolysaccharide.
 3. A method according to claim 1 whereinthe immunogen also comprises a T-cell epitope.
 4. A method according toclaim 1 wherein the immunogen further comprises a carrier protein.
 5. Amaterial according to claim 2 wherein the immunogen further comprises acarrier protein.
 6. The material according to claim 2, wherein theimmunogen further comprises a T-cell epitope.